Vaccines with enhanced immune response and methods for their preparation

ABSTRACT

The present invention is concerned with vaccines and their preparation. An effective long-term immune response, especially in mammals, can be produced using a vaccine comprising an antigen encapsulated in liposomes, a suitable adjuvant and a carrier comprising a continuous phase of a hydrophobic substance. The vaccine is particularly effective in eliciting the production of antibodies that recognize epitopes of native proteins.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationsU.S. Ser. No. 60/246,075 filed Nov. 7, 2000 and U.S. Ser. No. 60/307,159filed Jul. 24, 2001, the disclosures of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunology, in particular,to vaccines and their preparation.

BACKGROUND OF THE INVENTION

Generally, vaccines use low doses of a specific antigen to build upresistance in a host to the effects of larger doses of the antigen orsimilar antigenic compounds. Antigens used in vaccines are usually partsof whole organisms or denatured toxins (toxoids) that induce theproduction of antibodies. Unfortunately, only some of the antibodiesproduced bind to the target organism or toxin because, in most cases,the antigen used in the vaccine differs structurally from the target.The limited availability of useful antigens has posed limitations tovaccine development in the past. Advances in genetic engineering havemade the production of antigens by recombinant means possible. However,use of antigens produced by recombinant means often results in poorproduction of antibodies with poor affinity for the target nativeantigen for reasons given above. The effect of immunization can beenhanced when more antibodies with high affinity for their target areproduced. There is a need in the art to develop vaccines that produce anenhanced immune response without increasing the amount of antigen usedin the vaccine. Particularly, there is a need for single administrationvaccines that eliminate or reduce the need for booster immunizations.

Many immunization strategies would benefit from such development.Vaccines that use antigens derived from mammalian, viral, bacterial,fungal or yeast sources have many uses. For example, antigens fromviral, bacterial, fungal or yeast sources are useful in the preventionof disease. Antigens from mammals may be used in cancer therapy orimmunocontraception. Immunocontraceptive vaccines use mammalian derivedantigens that result in transient infertility or sterility of a host,particularly a mammalian host, by favouring the production of antibodieswith affinity for the oocyte surface. Immunocontraceptive vaccines finduse in the control of wild animal populations, including populations offeral domestic animals such as cats.

In particular, feral cat populations have been difficult to control andthreaten many birds and small animals. Stray feral cats also act asvectors for human and animal diseases. Various methods includinghunting, trapping and poisoning have been used in an effort to controlstray cat populations but these methods have met with limited successand with public opposition. Surgical sterilization of feral cats hasbeen increasingly used as a humane tool to lower feral cat populationsduring the last two decades. Acceptance of this procedure is widespread;however, disadvantages include cost, changes in behaviour and risk ofinfection and mortality. Despite the success of large-scale surgicalsterilization, such programs are not financially or logisticallyfeasible in many locations since capture of animals is time-consuming,difficult and stressful for the animal. Immunocontraception offers analternate procedure with lower costs and ease of administration.However, long-term immunocontraception generally requires boostervaccinations, making it impractical for the control of wild andfree-roaming species.

Vaccines generally comprise an antigen, which elicits the immuneresponse in the host, and a variety of carriers, excipients andadjuvants useful for administering the antigen to the host.

Liposomes, which encapsulate the antigen, have increasingly been used invaccine delivery. It has been shown that liposome delivery of denaturedantigens favours the production of antibodies that recognize nativeepitopes (Muttilainen, S., I. Idanpaan-Heikkila, E. Wahlstrom, M.Nurminen, P. H. Makela and M. Sarvas. 1995. “The Neisseria meningitidisouter membrane protein P1 produced in Bacillus subtilis andreconstituted into phospholipid vesicles elicits antibodies to native P1epitopes.” Microbial Pathogen. 18:423-436). While liposomes are usefulvaccine delivery vehicles, their use alone has not provided an effectivesingle dose vaccine, particularly with respect to immunocontraceptivevaccines.

Most immunocontraceptive vaccines use Freund's Complete Adjuvant (FCA)followed by Freund's Incomplete Adjuvant (FIA) in multiple injections toaid production of sufficient antibodies to have an immunocontraceptiveeffect (see Ivanova, et al., 1995. “Contraceptive potential of porcinezona pellucida in cats.” Theriogenology. 43:969-981 and Sacco et al.,1989. “Effect of varying dosage and adjuvants on antibody response insquirrel monkeys (Saimiri sciureus) immunized with the porcine zonapellucida Mr=55,000 glycoprotein (ZP3).” Am. J. Reprod. Immunol.21:1-8). Other adjuvants such as Ribi™ and TiterMax™ have been used bysome investigators. Alum (aluminum phosphate and/or hydroxide) has along history of use as an adjuvant. Alum is the only adjuvant recognizedas safe by the Food and Drug Administration. Many immunocontraceptivevaccines that use alum require a primary injection and several boosterinjections to produce sufficient antibodies for an immunocontraceptiveeffect (see Bagavant et al., 1994. “Antifertility effects of porcinezona pellucida-3 immunization using permissible adjuvants in femalebonnet monkeys (Macaca radiata): reversibility, effect on folliculardevelopment and hormonal profiles.” J. Reprod. Fertil. 102:17-25). Somestudies have shown that alum is not a suitable adjuvant for zonapellucida immunocontraceptive vaccines (see Sacco et al., 1989. Am. J.Reprod. Immunol. 21:1-8 and Bagavant et al., 1994. J. Reprod. Fertil.102:17-25).

Prior art has generally relied on the use of an aqueous medium oroil-in-water emulsions as carriers. For example, Muttilainen et al.(Microbial Pathogen. 18:423-436 (1995) use an aqueous medium incombination with liposomal delivery to elicit an immune response.Popescu (U.S. Pat. No. 5,897,873 issued Apr. 27, 1999 and U.S. Pat. No.6,090,406 issued Jul. 18, 2000), Alving (U.S. Pat. No. 6,093,406 issuedJul. 25, 2000 and U.S. Pat. No. 6,110,492 issued Aug. 29, 2000) andMuderhwa et al. (“Oil-in-water liposomal emulsions: Characterization andpotential use in vaccine delivery”, (December, 1999) J Pharm Sci.88(12):1332-9) also use liposomal systems together with an oil-in-watercarrier as the delivery system in a vaccine. Popescu uses alum withliposomes consisting of cholesterol esterified with succinate or otherorganic acids. U.S. Pat. No. 6,093,406 teaches the use of alum andliposomes comprising Lipid A or non-pyrogenic Lipid A in an oil-in-wateremulsion to deliver a vaccine based on malarial antigens. U.S. Pat. No.6,110,492 and Muderhwa teach the use of liposomes comprising Lipid A ornon-pyrogenic Lipid A in an oil-in-water emulsion to deliver prostratespecific antigens.

Commonly owned U.S. Pat. No. 5,736,141, issued on Apr. 7, 1998, teachesa single dose immunocontraceptive vaccine for seals derived from zonapellucida antigens. While the results achieved with this vaccine aregood, there is still a need for a single-dose, long lastingimmunocontraceptive vaccine effective in a variety of species usingadjuvants approved by the Food and Drug Administration.

There also remains a need for long lasting immunovaccines in generalwhich are effective using a variety of antigens in a variety of speciesusing adjuvants approved by the Food and Drug Administration.

SUMMARY OF THE INVENTION

In accordance with the invention, there is provided a composition foruse as a vaccine, comprising:

-   -   (a) a carrier comprising a continuous phase of a hydrophobic        substance;    -   (b) liposomes;    -   (c) an antigen; and,    -   (d) a suitable adjuvant.

There is further provided a method for potentiating an immune responsein an animal, which method comprises administering to the animal aneffective amount of a vaccine composition comprising:

-   -   (a) a carrier comprising a continuous phase of a hydrophobic        substance;    -   (b) liposomes;    -   (c) an antigen; and,    -   (d) a suitable adjuvant.

Still further there is provided a method of preparing a vaccinecomposition comprising the steps of:

-   -   (a) encapsulating an antigen or an antigen/adjuvant complex in        liposomes to form liposome-encapsulated antigen;    -   (b) mixing the liposome-encapsulated antigen with a carrier        comprising a continuous phase of a hydrophobic substance; and,    -   (c) adding a suitable adjuvant if an antigen/adjuvant complex is        not used in part (a).

Unexpectedly and uniquely, it has now been found that using a continuousphase of a hydrophobic substance as the carrier in a vaccine compositionof the present invention enhances the immune response. The enhancedresponse is characterized by long-lived high antibody titres following asingle vaccine administration resulting in enhanced duration of theimmune response. This is particularly true for vaccines that alsocomprise liposome-encapsulated antigen and an adjuvant (or mixture ofantigen/adjuvant). Vaccine compositions of the present invention aregenerally effective as a single dose providing a long-term immuneresponse in a variety of species, typically not requiring boosters.

DETAILED DESCRIPTION OF THE INVENTION

While not being held to any particular theory of action, it is thoughtthat, when a vaccine composition of the present invention is used, IgGantibody production occurs in two phases and the antibodies produced ineach phase differ in their epitope recognition. The antibodies producedin the second phase of IgG production have more affinity for nativeprotein antigens, thus making the vaccine more effective. Use ofconventional vaccines with a primary and booster injection producesantibodies having different binding specificity for an antigen than useof a vaccine composition of the present invention.

The carrier comprises a continuous phase of a hydrophobic substance,preferably a liquid hydrophobic substance. The continuous phase may bean essentially pure hydrophobic substance, a mixture of hydrophobicsubstances, an emulsion of water-in-a hydrophobic substance or anemulsion of water-in-a mixture of hydrophobic substances.

Hydrophobic substances that are useful in the present invention arethose that are pharmaceutically and/or immunologically acceptable.Ideally, the hydrophobic substance is one that has been approved for useby health regulatory agencies such as the U.S. Food and DrugAdministration. The carrier is preferably a liquid but certainhydrophobic substances that are not liquids at atmospheric temperaturemay be liquified, for example by warming, and are also useful in thisinvention.

Oil or water-in-oil emulsions are particularly suitable carriers for usein the present invention. Oils should be pharmaceutically and/orimmunologically acceptable. Preferred examples of oils are mineral oil(especially light or low viscosity mineral oil), vegetable oil (e.g.corn or canola oil), nut oil (e.g. peanut oil) and squalene. A lowviscosity mineral oil is most preferred. Animal fats and artificialhydrophobic polymeric materials, particularly those that are liquid atatmospheric temperature or that can be liquified relatively easily, mayalso be used.

The amount of hydrophobic substance used is not critical but istypically from about 0.1 ml per dose to about 1.5 ml per dose, dependingon the size of the animal and the amount of antigen being used. Forsmall animals, the amount of hydrophobic substance is preferably fromabout 0.20 ml to about 1.0 ml per dose, while for large animals, theamount is preferably from about 0.45 ml to about 1.5 ml per dose.Typically, 0.25 ml per dose is used for small animals while 0.5 ml perdose is used for large animals.

Suitable antigens are any chemicals that are capable of producing animmune response in a host organism. Preferably, the antigen is asuitable native, non-native, recombinant or denatured protein orpeptide, or a fragment thereof, that is capable of producing the desiredimmune response in a host organism. Host organisms are preferablyanimals (including mammals), more preferably cats, rabbits, horsesand/or deer. The antigen can be of a viral, bacterial, protozoal ormammalian origin. Antigens are generally known to be any chemicals(typically proteins or other peptides) that are capable of eliciting animmune response in a host organism. More particularly, when an antigenis introduced into a host organism, it binds to an antibody on B cellscausing the host to produce more of the antibody. For a generaldiscussion of antigens and the immune response, see Kuby, J., Immunology3^(rd) Ed. W.H. Freeman & C. NY (1997), the disclosure of which ishereby incorporated by reference.

Antigens that elicit an immune response related to cancer, contraceptionand other biological conditions or effects may be used in thepreparation of immunovaccines. Some typical, non-limiting examples ofantigens that may be used are alcohol dehydrogenase (ADH),streptokinase, hepatitis B surface antigen and zona pellucida (ZP)glycoproteins.

When the desired immune response is contraception in mammals, the targetepitopes are found on mammalian oocytes. Zona pellucida (ZP)glycoproteins or recombinant proteins or peptide fragments derivedtherefrom may be used in this case. In particular, heat extractedsolubilized isolated zona pellucida glycoproteins (SIZP) may be used asthe antigen in an immunocontraceptive vaccine. More particularly,soluble intact porcine zona pellucida may be used.

The amount of antigen used in a dose of the vaccine composition can varydepending on the type of antigen and the size of the host. One skilledin the art will be able to determine, without undue experimentation, theeffective amount of antigen to use in a particular application.

In the case of SIZP, the amount typically used falls in the range fromabout 15 μg to about 2 mg per dose. Preferably, the range is from about20 μg to about 2 mg per dose, more preferably from about 20 μg to about200 μg, and even more preferably from about 40 μg to about 120 μg.Typically, the amount for a small animal is about 50 μg per dose whilefor a large animal it is about 100 μg per dose.

In compositions of the present invention, antigens produce enhancedlevels of host antibodies that bind to native epitopes of the targetprotein. This is the case even though the antigen may be a non-native,recombinant or denatured protein or peptide, or a fragment thereof.While not wishing to be held to any particular theory, this may be dueto the antigen being held in a native-like three-dimensionalconformation in the liposomes.

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single bilayer membrane) or multilamellar vesicles(onion-like structures characterized by multimembrane bilayers, eachseparated from the next by an aqueous layer. A general discussion ofliposomes can be found in Gregoriadis G. (1990) Immunological adjuvants:A role for liposomes, Immunol. Today 11:89-97 and Frezard, F. (1999)Liposomes: From biophysics to the design of peptide vaccines. Braz. J.Med. Bio. Res 32:181-189, the disclosures of which are herebyincorporated by reference.

Although any liposomes may be used in this invention, includingliposomes made from archaebacterial lipids, particularly usefulliposomes use phospholipids and unesterified cholesterol in the liposomeformulation. The cholesterol is used to stabilize the liposomes and anyother compound that stabilizes liposomes may replace the cholesterol.Other liposome stabilizing compounds are known to those skilled in theart. The use of the particularly preferred liposomes may result inlimiting the electrostatic association between the antigen and theliposomes. Consequently, most of the antigen may be sequestered in theinterior of the liposomes.

Phospholipids that are preferably used in the preparation of liposomesare those with at least one head group selected from the groupconsisting of phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine and phosphoinositol. More preferred are liposomes thatcomprise lipids in phospholipon 90 G.

The amount of lipid used to form liposomes depends on the antigen beingused but is typically in a range from about 0.05 gram to about 0.5 gramper dose of vaccine. Preferably, the amount is about 0.1 gram per dose.When unesterified cholesterol is also used in liposome formulation, thecholesterol is used in an amount equivalent to about 10% of the amountof lipid. The preferred amount of cholesterol is about 0.01 gram perdose of vaccine. If a compound other than cholesterol is used tostabilize the liposomes, one skilled in the art can readily determinethe amount needed in the formulation.

In a more preferred aspect, the vaccine compositions of the presentinvention are essentially free from Lipid A, including non-pyrogenicLipid A. For the purposes of this specification, when the term Lipid Ais used, it is understood to encompass non-pyrogenic Lipid A as well.Lipid A is often found in liposomal formulations of the prior art. LipidA has many undesirable side-effects which may be overcome usingnon-pyrogenic Lipid A, but even then, Lipid A has many pharmaceuticalreactions other than the pyrogenic one and may still cause many adversereactions. It is therefore desirable to exclude Lipid A from thecompositions of this invention.

Suitable adjuvants are alum, other compounds of aluminum, Bacillus ofCalmette and Guerin (BCG), TiterMax™, Ribi™, Freund's Complete Adjuvant(FCA) and a new adjuvant disclosed by the United States Department ofAgriculture's (USDA) National Wildlife Research Center on their web siteat http://www.aphis.usda.gov/ws/nwrc/pzp.htm based on Johne's antigen.Alum, other compounds of aluminum, TiterMax™ and the new USDA adjuvantare preferred. Enhanced immune response is found even when the adjuvantis alum, which is surprising in view of the prior art (Sacco et al.1989. Am. J. Reprod. Immunol., 21:1-8). Alum is particularly preferredas the adjuvant.

Alum is generally considered to be any salt of aluminum, in particular,the salts of inorganic acids. Hydroxide and phosphate salts areparticularly useful as adjuvants. A suitable alum adjuvant is sold underthe trade name, ImjectAlum™ (Pierce Chemical Company) that consists ofan aqueous solution of aluminum hydroxide (45 mg/ml) and magnesiumhydroxide (40 mg/ml) plus inactive stabilizers. Alum is a particularlyadvantageous adjuvant since it already has regulatory approval and it iswidely accepted in the art.

The amount of adjuvant used depends on the amount of antigen and on thetype of adjuvant. One skilled in the art can readily determine theamount of adjuvant needed in a particular application. Forimmunocontraception, a suitable quantity of ImjectAlum™ for a rabbit is0.1 ml/dose of vaccine, whereas, a suitable quantity of ImjectAlum™ fora horse is 0.5 ml/dose.

The vaccine composition is generally formulated by: encapsulating anantigen or an antigen/adjuvant complex in liposomes to formliposome-encapsulated antigen and mixing the liposome-encapsulatedantigen with a carrier comprising a continuous phase of a hydrophobicsubstance. If an antigen/adjuvant complex is not used in the first step,a suitable adjuvant may be added to the liposome-encapsulated antigen,to the mixture of liposome-encapsulated antigen and carrier, or to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen. The order of the process may depend on the type of adjuvantused. Typically, when an adjuvant like alum is used, the adjuvant andthe antigen are mixed first to form an antigen/adjuvant complex followedby encapsulation of the antigen/adjuvant complex with liposomes. Theresulting liposome-encapsulated antigen is then mixed with the carrier.(It should be noted that the term “liposome-encapsulated antigen” mayrefer to encapsulation of the antigen alone or to the encapsulation ofthe antigen/adjuvant complex depending on the context.) This promotesintimate contact between the adjuvant and the antigen and may, at leastin part, account for the surprisingly good immune response when alum isused as the adjuvant. When another is used, the antigen may be firstencapsulated in liposomes and the resulting liposome-encapsulatedantigen is then mixed into the adjuvant in a hydrophobic substance.

In formulating a vaccine composition that is substantially free ofwater, antigen or antigen/adjuvant complex is encapsulated withliposomes and mixed with a hydrophobic substance. In formulating avaccine in an emulsion of water-in-a hydrophobic substance, the antigenor antigen/adjuvant complex is encapsulated with liposomes in an aqueousmedium followed by the mixing of the aqueous medium with a hydrophobicsubstance. In the case of the emulsion, to maintain the hydrophobicsubstance in the continuous phase, the aqueous medium containing theliposomes may be added in aliquots with mixing to the hydrophobicsubstance.

In all methods of formulation, the liposome-encapsulated antigen may befreeze-dried before being mixed with the hydrophobic substance. or withthe aqueous medium as the case may be. In some instances, anantigen/adjuvant complex may be encapsulated by liposomes followed byfreeze-drying. In other instances, the antigen may be encapsulated byliposomes followed by the addition of adjuvant then freeze-drying toform a freeze-dried liposome-encapsulated antigen with externaladjuvant. In yet another instance, the antigen may be encapsulated byliposomes followed by freeze-drying before the addition of adjuvant.Freeze-drying may promote better interaction between the adjuvant andthe antigen resulting in a more efficacious vaccine.

Formulation of the liposome-encapsulated antigen into a hydrophobicsubstance may also involve the use of an emulsifier to promote more evendistribution of the liposomes in the hydrophobic substance. Typicalemulsifiers are well-known in the art and include mannide oleate(Arlacel™ A), lecithin, Tween™ 80, Spans™ 20,80, 83 and 85. Mannideoleate is a preferred emulsifier. The emulsifier is used in an amounteffective to promote even distribution of the liposomes. Typically, thevolume ratio (v/v) of hydrophobic substance to emulsifier is in therange of about 5:1 to about 15:1 with a ratio of about 10:1 beingpreferred.

Administration of the vaccine composition can be done by any convenientmethod and will depend on the antigen being used. Vaccine compositionsmay be administered parenterally (including intramuscularly,sub-cutaneously) or rectally. Parenteral administration is preferred.

For parenteral application, particularly convenient unit dosage formsare ampoules. Techniques that deliver the vaccine by injection and byremote delivery using darts, spring loaded syringes with jab sticks,air/carbon dioxide powered rifles, Wester gun and/or Ballistivet™biobullets and retain the biological activity are particularlypreferred.

The amount of vaccine composition administered to a host may depend onthe amount of antigen used in a dose and on the effective amount ofantigen required for a particular application. In the case of SIZP, thesize of each dose administered to an animal is typically from about 0.25ml to about 2.0 ml depending on the size of the animal. For smalleranimals (for example, cats, rabbits, etc.) the size of the dose istypically about 0.5 ml while for larger animals (for example, horses,fallow-deer, white-tail deer, etc.) the size of the dose is typicallyabout 1.0 ml. Typically, even when the amount of SIZP is varied, thedose size is kept fairly constant.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of non-limiting exampleshaving regard to the appended drawing in which:

FIG. 1 is a diagram showing the position of recombinant ZPB1 and ZPB2;ZPC1 and ZPC2 of ZPB and ZPC proteins of porcine zona pellucida thatwere generated in the pRSET vectors.

EXAMPLES Example 1 Preparation of the Vaccine Composition

The vaccine composition can be formulated to be water-free or to containvarious quantities of water (by using an aqueous medium, for example,saline, phosphate buffered saline (PBS) or pyrogen-free water) whilemaintaining a continuous oil phase. Procedure 1 described below appliesto the water-free formulation of the vaccine composition. Procedure 2described below applies to the water containing formulation of thevaccine composition, that is, the water-in-oil emulsion. The twoprocedures can also vary depending on the adjuvant being used. Asexamples, method A applies to formulations of the vaccine compositioncontaining alum and method B applies to formulations containing Freund'sComplete Adjuvant (FCA). Other adjuvants may be accommodated by adaptingeither method A or method B. The procedures described below incorporateporcine soluble intact zona pellucida (SIZP) as antigen, other antigenscan replace SIZP in the formulation. For example, alcohol dehydrogenase(ADH), streptokinase or hepatitis B surface antigen can also be used asthe antigen.

Procedure 1: Water-free Formulation.

Method A. Alum Adjuvant.

SIZP is prepared as previously described (Brown, R. G., W. D. Bowen, J.D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B. Pohajdak.(1997) Temporal trends in antibody production in captive grey seals,harp and hooded seals to a single administration immunocontraceptivevaccine. J. Reproductive Immunology 35:53-64). The quantity of SIZPneeded for the number of doses of the vaccine being prepared is weighed(the usual quantity of SIZP used for immunization is 50 μg for smallanimals and 100 μg for large animals). The SIZP is dissolved inpyrogen-free distilled water to give a final concentration of 2 mg/ml.An equal volume of ImjectAlum™ (an alum product from Pierce ChemicalCo., catalogue # 77161) is added and the suspension is mixed, thenfreeze-dried.

To form liposomes, phospholipon 90 G (or other lipids selected fromphosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine,phosphoinositol, archaebacterial lipids, without limitation, that form aclosed lipid bilayer containing an entrapped antigen) is weighed (0.1g/dose of the vaccine composition). The phospholipon 90 G is mixed withcholesterol (0.01 g/dose of vaccine composition) and the mixture isdissolved in chloroform:methanol (1/1;v/v; 1.5 ml/dose of the vaccinecomposition). Cholesterol can be replaced with other compounds thatstabilize liposomes at concentrations determined by those skilled in theart. Washed glass beads (approximately 3 mm in diameter; 15 ml for 10doses of the vaccine) are added and the mixture is evaporated underreduced pressure using a rotary evaporator until free ofchloroform:methanol. To ensure removal of all chloroform:methanol, themixture is placed in a dessicator under reduced pressure overnight atroom temperature.

The freeze-dried SIZP/alum complex is suspended in pyrogen-freedistilled water (5 ml/mg SIZP) and the suspension added to the flaskcontaining the mixture of phospholipon 90 G/cholesterol coating theflask and glass beads. The contents of the flask are allowed to standwithout agitation for 30 minutes. After 30 minutes, the flask is placedin a water bath at 35-40° C. and stirred gently with a spatula to formthe liposomes. A microscope is used to evaluate liposome formation andstirring is continued with increased shaking until the mixture containspredominately multilamellar liposomes recognized by those skilled in theart. The liposomes are freeze-dried and the resulting freeze-driedliposomes are suspended in low viscosity mineral oil (0.25 ml oil/dosefor small animals and 0.5 ml oil/dose for large animals) containingmannide oleate as an emulsifier (10:1:oil:emulsifier:v/v). Sinceliposomes are suspended in oil and are not in solution, it is necessaryto determine if the procedures used result in an even distribution ofSIZP in each dose. To determine if freeze-dried liposomes containingSIZP are equally distributed in oil, SIZP is labelled with ¹⁴C byreductive methylation (Jentoft, N. and D. G. Dearborn. 1979. Labellingof proteins by reductive methylation using sodium cyanoborohydride. J.Biol. Chem. 254:4359-4365, the disclosure of which is herebyincorporated by reference) and the radioactive SIZP used to prepare twopreparations of the vaccine. Individual doses of the vaccine areprepared and radioactivity in each dose determined, thereby determiningthe content of SIZP in each dose of the vaccine (Table 1). Thedistribution of SIZP in each dose of the vaccine is highly reproducible(standard deviation, SD, was less than +/−10%). TABLE 1 Distribution of¹⁴C-labelled SIZP in doses of the vaccine from two preparations SamplePreparation 1 Preparation 2 No. μg SIZP/dose μg SIZP/dose 1 68 55 2 6653 3 57 53 4 65 62 5 56 59 6 51 57 7 62 58 8 55 60 9 69 51 10 54 59 1152 57 12 61 61 13 64 61 14 61 60 15 — 56 Average 60 57 Standard 5.9 3.2DeviationMethod B. FCA Adjuvant.

Preparation of the vaccine composition to contain FCA as a adjuvant inplace of alum, is similar to method A, except SIZP by itself, ratherthan as a SIZP/alum complex, is encapsulated in liposomes as describedabove. The liposomes containing SIZP are freeze-dried, and thefreeze-dried liposomes are added to FCA in aliquots with mixing topromote an equal distribution of liposomes in the oil. The resultingsuspension of freeze-dried liposomes containing SIZP in FCA isadministered to animals being vaccinated.

Procedure 2. Water-Containing Formulation.

Method A. Alum Adjuvant.

SIZP is prepared as previously described (Brown, R. G., W. D. Bowen, J.D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B. Pohajdak.(1997) Temporal trends in antibody production in captive grey seals,harp and hooded seals to a single administration immunocontraceptivevaccine. J. Reproductive Immunology 35:53-64, the disclosure of which ishereby incorporated by reference). The quantity of SIZP needed for thenumber of doses of the vaccine being prepared is weighed (the usualquantity of SIZP used for immunization is 50 μg for small animals and100 μg for large animals). The SIZP is dissolved in pyrogen-freedistilled water to give a final concentration of 2 mg/ml. An equalvolume of ImjectAlum™ (an alum product from Pierce Chemical Co.,catalogue # 77161) is added and the suspension is mixed, thenfreeze-dried.

To form liposomes, phospholipon 90 G (or other lipids selected fromphosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine,phosphoinositol, etc. that form a closed lipid bilayer containing anentrapped aqueous volume) is weighed (0.1 g/dose of the vaccinecomposition). The phospholipon 90 G is mixed with cholesterol (0.01g/dose of the vaccine composition) and the mixture is dissolved inchloroform:methanol (1/1;v/v; 1.5 ml/dose of the vaccine composition).Cholesterol can be replaced with other compounds that stabilizeliposomes at concentrations determined by those skilled in the art.Washed glass beads (approximately 3 mm in diameter; 15 ml for 10 dosesof the vaccine) are added and the mixture is evaporated under reducedpressure using a rotary evaporator until free of chloroform:methanol. Toensure removal of all chloroform:methanol, the mixture is placed in adessicator under reduced pressure overnight at room temperature.

The freeze-dried SIZP/alum complex is suspended in saline (5 ml/mg SIZP)and the suspension is added to the flask containing the mixture ofphospholipon 90 G/cholesterol coating the flask and glass beads. Thecontents of the flask are allowed to stand without agitation for 30minutes. After 30 minutes, the flask is placed in a water bath at 35-40°C. and stirred gently with a spatula to form the liposomes. A microscopeis used to evaluate liposome formation and stirring is continued withincreased shaking until the mixture contains predominately multilamellarliposomes recognized by those skilled in the art. The aqueous suspensionof liposomes (0.25 ml/dose for small animals and 0.5 ml/dose for largeanimals) is added to low viscosity mineral oil (0.25 ml oil/dose forsmall animals and 0.5 ml oil/dose for large animals) containing mannideoleate as an emulsifier (10:1:oil:emulsifier:v/v). The aqueoussuspension of liposomes is added to the low mineral oil phase inaliquots with mixing between aliquots to maintain the continuous oilphase.

Method B. FCA Adjuvant.

Preparation of the vaccine composition to contain FCA as adjuvant inplace of alum, is similar to method A, except SIZP by itself, ratherthan as a SIZP/alum complex, is encapsulated in liposomes as describedabove. The aqueous suspension of liposomes is added to FCA in aliquotswith mixing between aliquots to maintain the continuous oil phase. Theresulting aqueous suspension of liposomes containing SIZP in FCA isadministered to animals being vaccinated.

Note: In some trials, the quantity of SIZP is varied to study theresponse of immunized animals to different quantities of antigen. Insuch experiments, the volume of the vaccine administered to smallanimals (cats, rabbits, etc.) was 0.5 ml and the volume administered tolarge animals (horses, fallow deer, white-tailed deer, etc.) was 1.0 ml.In such cases, the quantity of liposomes in each dose of the vaccine ismaintained constant while the quantity of antigen encapsulated inliposomes varied.

Example 2 Immunization of Rabbits Against Native and Denatured YeastAlcohol Dehydrogenase (ADH)

The vaccine composition is unique in producing high titers of anti-SIZPantibodies that are long-lasting following a single administration. Todetermine if the vaccine composition would produce high antibody titerswith other antigens, particularly proteins that are not bound to cellmembranes, rabbits are immunized with yeast alcohol dehydrogenase (ADH).Two forms of ADH are used as antigen, namely, native ADH and ADH thathad been treated to denature the protein. To denature ADH, ADH istreated with mercaptoethanol (10% v/v in Tris buffer, 0.1 M, pH 7.5, 30min, 100° C.). The solution is dialyzed against distilled water andfreeze-dried. Four rabbits (2 for each treatment) are immunized withnative or denatured ADH (40 μg) using a primary injection with Freund'scomplete adjuvant (FCA) followed by a booster injection with Freund'sincomplete adjuvant (FIA) given one month later. The post-immunizationperiod is considered to have begun after the booster injection. At thesame time as the booster injection is administered, four rabbits (2 foreach treatment) are immunized by single administration with eithernative or denatured ADH (40 μg) using the vaccine composition, that isADH is encapsulated in liposomes that are suspended in saline (0.5 ml)and emulsified in FCA (0.5 ml). Anti-ADH titers are measured by ELISAusing both native and denatured ADH (Table 2).

When rabbits are immunized with native ADH, the resulting serumcontained similar quantities of anti-ADH antibodies when native ADH isdelivered with the vaccine composition or by using a primary injectionwith one booster. In contrast, serum from rabbits immunized withdenatured ADH delivered with the vaccine composition contain 2.7 timesmore antibody that bound to native ADH than serum from rabbits that areimmunized with denatured ADH with a primary injection and one booster(P<0.01; T=4.14; df=6). In all cases, titers are higher in rabbitsimmunized with native ADH than when rabbits were immunized withdenatured ADH. This indicates that native ADH is a better antigen thandenatured ADH. Since many protein antigens are denatured to some degreeduring extraction and isolation or when produced by recombinant means,increased production of antibodies that bind better to native proteinscan significantly improve the outcome of vaccination as demonstrated byimmunocontraception of a variety of mammals with SIZP using the vaccinecomposition of the present invention.

Furthermore, anti-ADH sera from rabbits 237 and 238 recognize denaturedADH in Western blots with about 4-5 times the intensity of anti-ADH serafrom rabbits 235 and 236. This confirms the results of titermeasurements indicating that immunization of rabbits with the vaccinecomposition favours the production of anti-ADH antibodies that bindbetter to native ADH since many proteins are known to refold during theWestern protocol to a more native state. This conclusion is supported byMuttilainen et al. (1995) in a study of Neisseria meningitidis outermembrane protein P1, who found antibodies to native P1 were elicited inmice vaccinated with denatured P1 constituted into phospholipid vesicles(liposomes). However, Muttilainen et al. (1995) did not use oil in theirvaccine formulation, therefore, their immunization protocol wasdifferent than the present invention. TABLE 2 Production of anti-ADHantibodies by rabbits immunized with native or denatured ADH deliveredwith and without liposome encapsulation Anti-ADH titer Immunization (%of reference serum)¹ Rabbit Native ADH³ Denatured ADH³ ID No. AntigenDelivery² 4⁴ 5⁴ 4⁴ 5⁴ 231 native −Liposomes 187 148 19 23 ADH 232 native−Liposomes 200 161 17 15 ADH 233 native +Liposomes 239 156 21 14 ADH 234native +Liposomes 100 100 19 11 ADH 235 denatured −Liposomes 41 37 7 3ADH 236 denatured −Liposomes 30 18 7 4 ADH 237 denatured +Liposomes 10171 8 3 ADH 238 denatured +Liposomes 101 63 12 7 ADH¹Anti-ADH serum from rabbit 234 is used as the reference serum.²Native and denatured ADH are administered with the vaccine composition,that is encapsulated in liposomes with FCA as a single i.m. injection(+liposomes) or suspended in FCA followed one month later as a boosterinjection using FIA (−liposomes). Rabbits receive 40 μg ADH with eachadministration.³ADH is purchased from Sigma Chemical Co. Denatured ADH is produced bytreating native ADH with mercaptoethanol and heating to 100° C. for 30minutes. The resulting denatured ADH contain three major proteins havingmolecular weights of 26, 33 and 40 kDa. Titers are measured by ELISAusing native and denatured ADH as antigen to coat ELISA plates inseparate determinations.⁴Post-immunization in months.Note:The reference serum noted in Table 2 is rabbit serum ID No. 234.

Example 3 Immunocontraception of Rabbits

Sera from rabbits immunized with a placebo vaccine that contained allingredients of the vaccine composition except the antigen (porcine SIZP)contain no anti-porcine SIZP antibodies (see Table 3A). Immunization ofrabbits with porcine SIZP (40 μg) encapsulated in liposomes containingphosholipon 90 G (0.1 g), cholesterol (0.01 g) in saline (0.5 ml)emulsified in FCA adjuvant (0.5 ml) produce high titers of anti-SIZPantibodies during the 12 month post-immunization monitoring periodfollowing a single administration of the vaccine. Immunization ofrabbits with porcine SIZP (40 μg) encapsulated in liposomes with MF 59adjuvant (0.5 ml) produce low anti-SIZP titers. In contrast,immunization of rabbits with porcine SIZP encapsulated in liposomes withalum adjuvant (100 μl, Pierce ImjectAlum™) produce anti-porcine SIZPtiters that are less than titers produced using FCA in earlypost-immunization but the titers are less different than between thealum and FCA runs by the 12^(th) month of post-immunization. Breeding ofrabbits established that a single administration of the vaccine usingFCA or alum reduces fertility of rabbits by 79 and 74% respectively(Table 3B). Immunization of rabbits by a single injection of SIZP (40μg) that is not encapsulated in liposomes with Gerbu adjuvant produceslow anti-porcine SIZP titers (Table 3A). As expected based on anti-SIZPtiters, rabbits immunized with SIZP that are not encapsulated inliposomes with Gerbu adjuvant have the same fertility as rabbits thatreceive the placebo vaccine (Table 3B). These results indicate thatvaccines comprising liposome-encapsulated antigen produce good resultsand that FCA and alum, particularly alum, are especially good adjuvants.TABLE 3A Effect of adjuvants on the production of anti-porcine SIZPantibodies by rabbits¹ Post-immunization anti-porcine SIZP titer (% ofreference ID Time (months) No. 0 1 2 3 4 5 6 7 11 12 Placebo 2 0 1 0 0 00 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 FCA 3 0 145 11294 82 45 23 24 20 20 15 0 100 71 68 83 19 29 26 18 25 16 0 125 111 122128 111 74 92 75 63 MF 59 1 0 3 1 1 1 2 0 ND ND ND 7 0 10 9 3 3 5 2 NDND ND 19 0 12 6 3 3 3 3 ND ND ND 20 0 37 21 13 15 13 10 ND ND ND Alum 110 19 12 12 11 14 18 7 8 10 12 0 20 10 10 8 6 11 5 2 4 23 0 31 16 40 2220 33 28 37 28 24 0 36 21 40 27 24 40 36 34 34 Gerbu without liposomeencapsulation of SIZP 9 0 5 2 1 1 1 1 1 ND ND 10 0 11 3 3 2 2 3 1 ND ND21 0 17 4 6 19 5 6 2 ND ND 22 0 24 3 6 3 4 4 7 ND ND¹Rabbits receive a single administration of the placebo vaccine, vaccinethat contained porcine SIZP (40 μg) encapsulated in liposomes (0.25 ml)with either FCA, MF 59 or alum adjuvants (0.25 ml) or vaccine thatcontained porcine SIZP (40 μg) dissolved in saline (0.25 ml) with gerbuadjuvant (0.25 ml).ND = not determined.

The reference serum used is from a rabbit immunized with porcine zonapellucida using a primary injection with Freund's complete adjuvant and2 booster injections with Freund's incomplete adjuvant. TABLE 3B Effectof adjuvants on the fertility of rabbits immunized against porcine SIZPLive births/mating¹ Average live % reduction in ID NO 1 2 3births/mating fertility Placebo 2 6 0 4 5.1 0 13 7 1 10 14 6 7 5 FCA 3 00 0 1.1 79 15 0 8 5 16 0 NM 0 MF 59 1 11 NM 10 6.1 0 7 0 0 NM 19 5 11 1120 6 0 7 Alum 11 0 0 6 1.3 74 12 0 0 0 23 0 0 0 24 3 5 2 Gerbu withoutliposome encapsulation 9 0 0 11 5.3 0 10 0 0 0 21 8 9 11 22 10 7 11NM = paired repeatedly with males without a successful mating¹Live births following a successful mating. The mating intervals were 65± 10, 141 ± 14 and 216 ± 14 days post-immunization for matings 1, 2 and3 respectively.

Example 4 Immunocontraception in Cats

Twenty-nine specific pathogen free domestic short hair cats are housedat the specific pathogen free facility at the University of Floridaunder the supervision of Dr. Julie Levy and Mr. Shawn Gorman. Estruscycling is monitored by daily observation and vaginal cytology. Vaccinecompositions of the present invention, placebo vaccines and serumsamples are coded as part of a double-blind study. The cats are dividedrandomly into 3 groups of nine or ten cats each. One group receives aplacebo vaccine that contains all components of the vaccine compositionexcept the antigen (porcine SIZP) by intramuscular injection. Each catin this group receives liposomes containing no antigen in saline (0.25ml) suspended in FCA (0.25 ml). Each cat in a second group of nine catsis immunized by intramuscular injection with the vaccine compositioncontaining SIZP (135 μg) encapsulated in liposomes in saline (0.25 ml)and suspended in FCA (0.25 ml). Each cat in a third group of nine catsis immunized by intramuscular injection with the vaccine compositioncontaining porcine SIZP (200 μg) with alum (0.12 ml, Pierce ChemicalCo., catalogue number 77161) encapsulated in liposomes in saline (0.12ml) and suspended in a suitable pharmacological carrier. Production ofanti-SIZP antibodies in cats is measured by ELISA using proteinA/alkaline phosphatase (Brown, R. G., W. D. Bowen, J. D. Eddington, W.C. Kimmins, M. Mezei, J. L. Parsons, B. Pohajdak. (1997) Temporal trendsin antibody production in captive grey seals, harp and hooded seals to asingle administration immunocontraceptive vaccine. J. ReproductiveImmunology 35:53-64).

A single administration of the vaccine composition using FCA producesanti-SIZP antibody titers that reached maximal titers within 2 months(Table 4). The average two months post-immunization titer is 58±2% ofthe reference serum which decreased to 41±4% of the reference serum atfour months post-immunization when a proven male cat is introduced tothe colony. Cats that receive a single administration of the vaccinecomposition using alum as adjuvant produce anti-SIZP antibodies with anaverage titer of 67±2% of the reference serum two monthspost-immunization.

Monthly serum samples from cats that are immunized with the placebovaccine containing all components of the vaccine composition except theantigen, have an average anti-SIZP titer of 0.6±0.2% of the referenceserum during the post-immunization monitoring period. Therefore, it isapparent that cats that received the placebo vaccine will producekittens during the post-immunization period. TABLE 4 Production ofanti-SIZP antibodies by cats immunized with the vaccine composition ofthe invention Anti-SIZP titer (% of reference serum) Cat IDPost-immunization (months) No. 0 1 2 3 4 5 6 7 8 9 10 11 Placebo 3 0 0 00 1 0 0 0 0 7 4 2 4 0 0 0 0 2 0 0 3 1 2 2 0 8 0 3 0 1 0 0 0 1 1 2 2 0 151 0 1 0 0 0 0 2 1 ND ND ND A 0 0 3 0 1 0 0 0 0 2 2 0 B 0 0 0 1 1 1 0 ND2 2 0 0 E 0 1 1 1 0 0 0 2 5 ND ND ND F 0 1 1 0 0 0 0 2 0 ND ND ND I 0 10 1 1 0 0 0 2 0 0 ND N 0 1 0 0 1 1 1 2 1 ND ND ND Vaccine with FCA 1 060 57 68 46 56 23 46 15 40 23 23 2 0 60 53 58 44 25 23 69 46 68 70 62 90 47 56 41 56 20 58 78 103 74 86 ND 13 0 42 64 53 66 55 47 29 33 12 16ND 14 0 48 51 30 45 40 10 20 9 10 12 7 C 0 56 54 49 34 21 16 12 14 24 1416 D 0 58 60 67 59 52 32 19 48 ND ND ND L 0 47 61 40 28 16 46 37 59 7059 72 G 0 59 65 79 85 81 92 94 96 115 152 79 O 0 48 42 53 42 24 26 28 2531 16 10 Vaccine with alum 1P 1 60 60 70 46 47 25 18 41 26 6 ND 1S 0 7356 43 24 42 19 18 18 14 4 ND 1T 0 62 62 58 30 34 14 12 8 11 4 ND 1V 2 7068 60 40 36 12 12 ND ND ND ND 1Y 1 84 82 84 81 80 67 34 44 28 ND ND 1Z 077 75 71 54 72 54 26 35 21 9 ND Z1 0 61 74 95 83 100 98 92 70 42 ND NDZ2 0 77 79 63 34 50 41 28 18 11 ND ND Z3 0 65 72 61 55 30 35 18 20 6 NDND Z4 0 73 ND 68 53 29 32 17 12 13 ND NDND = not determined

Example 5 Immunocontraception of Deer

Forty-one fallow deer (Dama dama) does on James Island, a 360-hectareisland that lies off the coast of southern British Columbia, areimmunized with the vaccine composition using FCA as adjuvant. Anothergroup of forty fallow deer does are immunized with the vaccinecomposition using alum as the adjuvant. For capture, the deer are baitedinto a large (200×200 meter) pen that is connected to a series of fencedenclosures and a raceway that terminates in a small building. Beforeimmunization, each deer is physically restrained and given a numberedear tag, a colored plastic collar or radio collar with a mortalitysensor, and a PIT (permanent identification transponder) tag bearing aunique code. Thus, if a treated deer loses all external marks, it couldstill be recognized as a treated animal from injury resulting from lossof ear tag and as a particular deer from the PIT tag. Each captive doeis injected intramuscularly in the rump with SIZP (100 μg) encapsulatedin liposomes with FCA or alum adjuvants. Untreated does serve ascontrols.

Anti-SIZP titers are measured as previously described (Brown, R. G., W.D. Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B.Pohjajdak. (1997) Temporal trends in antibody production in captive greyseals, harp and hooded seals to a single administrationimmunocontraceptive vaccine. J. Reproductive Immunology 35:53-64) exceptthat protein G/alkaline phosphatase replaced protein A/alkalinephosphatase since protein G has a higher affinity for fallow deerimmunoglobulin than does protein A. Relative to the affinities ofprotein A and protein G for rabbit immunoglobulin (the reference serum),the affinities of protein A and protein G for fallow deer immunoglobulinare 8 and 89% respectively. Fallow deer anti-SIZP titers are uncorrectedfor relative affinity of protein G (Table 5A). None of the does examined2 months or more following the rut and 8-9 months after being immunizedwith the vaccine containing FCA were pregnant, while 96% (192/200)untreated does are pregnant. Pregnancy is determined by examination ofthe reproductive tract for signs of pregnancy or by analyzing blood fromlive captured does for pregnancy-specific protein B (PSPB) byBioTracking, Inc. of Moscow, Id. (Willard et al., “Pregnancy detectionand the effects of age, body weight, and previous reproductiveperformance on pregnancy status and weaning rates of farmed fallow deer(Dama dama). J. Animal Science. 77:32-38 (1999), the disclosure of whichis hereby incorporated by reference). The contrast between the pregnancyrates of immunized and unimmunized does shows clearly that the vaccinecomposition containing FCA is effective in preventing conception. Sincethis is a multiple year study, as many as possible of the does are livecaptured. TABLE 5A Production of anti-SIZP antibodies by fallow deerimmunized with the vaccine composition of the invention containing FCAadjuvant. Post-immunization anti-SIZP titer (% of reference serum)Fallow deer Time (months) ID No. 0 1-2 7-10 Controls 99023 0 0 0 99024 00 0 99025 0 0 0 99027 0 0 0 99028 0 0 0 99029 0 0 0 Vaccine with FCA99026 0 117 ND 99025 0 72 ND 2000-06 0 96 ND 2000-08 0 94 ND 99007 0 ND36 99008 0 ND 60 99009 0 ND 94 99016 0 ND 60 99017 0 ND 66 99019 0 ND133 99020 0 ND 56ND = not determined

Other experiments were performed on white-tailed deer. None of thewhite-tailed deer immunized with the vaccine composition comprising FCAbecame pregnant one year post-immunization. Only one of the white-taileddeer immunized with the vaccine composition comprising alum did notbecome pregnant one year post-immunization. The results for anti-SIZPtiter levels are shown in Table 5B. TABLE 5B Production of anti-SIZPantibodies by white-tailed deer immunized with a composition of thepresent invention. White- tailed Anti-SIZP (% of reference serum) deerID Post-immunization (months) No. 0 2 4 5 8 12 Freund's completeadjuvant (FCA) 19 0 ND ND 85 ND 21 0 ND ND 139 ND 33 0 ND ND 125 ND Alum949 1 12 11 ND 48 27 916 1 6 4 ND 5 2 744 3 105 111 ND 123 75 694 0 7 7ND 5 4 956 0 5 4 ND 2 2 9 0 ND ND 3 ND ND 14 0 ND ND 8 ND ND 17 0 ND ND4 ND ND 27 0 ND ND 4 ND NDND = not determined

Example 6 The Effect of Oil Content on the Production of Anti-SIZPAntibodies

The vaccine composition yields good antibody titers following a singleadministration of an antigen, therefore, unless stated otherwise alltiters reported in the following example results from a singleadministration of the antigen in the vaccine formulation and otherimmunization protocols.

To determine if an aqueous phase is a necessary component of the vaccinecomposition to obtain a good immune response, three groups of rabbits (2or 3 rabbits/group) are immunized with three different preparations ofthe vaccine containing SIZP (50 μg SIZP/rabbit) encapsulated inliposomes that are suspended in saline (0.5 ml) and emulsified inFreund's complete adjuvant (0.5 ml). The proportion of oil phase andwater phase is equal in these preparations (Table 6A). TABLE 6A Effectof oil content of the vaccine composition on the production of anti-SIZPantibodies by rabbits Anti-SIZP titer (% of reference serum)¹ Oilcontent Post-immunization (months) (%, v/v) 0 1 2 3 4 50² 0 120 131 10324 0 38 38 24 17 50² 0 91 91 54 67 0 46 112 143 112 50² 0 54 65 51 ND 095 94 81 ND 0 47 49 32 ND 100³ 0 61 75 136 34 0 14 24 100 95 100⁴ 0 159149 215 27 0 50 196 244 128 100⁴ 0 11 30 48 29 0 15 28 40 41 100⁴ 0 5454 90 91 0 14 2 19 19 0 47 49 67 76¹Each line presents titers of blood samples taken from the same rabbit.²Liposomes containing SIZP (50 μg/rabbit) were suspended in saline (0.5ml) and this aqueous phase was emulsified in Freund's complete adjuvant(0.5 ml).³Liposomes containing SIZP (50 μg/rabbit) were freeze-dried and theresulting freeze-dried liposomes suspended in Freund's complete adjuvant(0.5 ml).⁴Liposomes containing SIZP (50 μg/rabbit) are freeze-dried and theresulting freeze-dried liposomes suspended in Freund's complete adjuvant(0.2 ml).ND = not determined.The vaccine formulated to contain no water is used to immunize fourgroups of rabbits (2 or 3 rabbits/group) with four differentpreparations of the vaccine containing SIZP (50 μg SIZP/rabbit)encapsulated in freeze-dried liposomes suspended in Freund's completeadjuvant (0.2 ml or 0.5 ml). Since Freund's complete adjuvant containsno water, these preparations are water-free and contained only an oilphase. Average anti-SIZP titers 4 months post-immunization are 55±44%(coefficient of variation, cv 80%) for rabbits that are immunized withthe composition containing 50% oil and 59±41% (cv 69%) for rabbits thatare immunized with the vaccine containing 100% oil. There is nodifference in response of female rabbits that received the vaccine with100% oil and female rabbits that are immunized with the vaccinecontaining 50% oil (P=0.87; F(1,45)=0.03; average titers were 71±7% for50% oil and 72±12% for 100% oil). These results indicate that thepresence of an aqueous phase is not necessary for a good immune responseto the vaccine.

To determine if there is a difference in duration of anti-SIZP titers inrabbits that are immunized with the vaccine composition with 50% and100% oil, anti-SIZP titers are measured for 12 months (Table 6B).Anti-SIZP titers during the 12 month post-immunization period aresimilar in rabbits immunized with 50% oil formulation and the rabbitimmunized with 100% oil formulation. To verify the biological effect ofimmunization with SIZP, proven female rabbits immunized with bothformulations of the vaccine are mated with proven males 3 times duringthe 12 month post-immunization period. Reduction in fertility was 80%for the rabbits that are immunized with the vaccine containing 50% oiland the female rabbit that is immunized with the vaccine containing 100%oil produce no offspring indicating that the biological effect ofreduced fertility is similar with both formulations of the vaccine.TABLE 6B Effect of oil content of the vaccine composition on theduration of anti-SIZP antibodies in rabbits Anti-SIZP titer (% ofreference serum) Rabbit Post-immunization (months) ID No. 0 1 2 3 4 5 67 11 12 50% oil content 3 0 145 112 94 82 45 23 24 20 20 15 0 100 71 6883 19 29 26 18 25 16 0 125 111 122 128 111 74 92 75 63 100% oil content1 0 127 138 ND ND ND 33 51 17 27ND = not determined.

Since liposomes are composed of material that is lipophilic, storage ofliposomes in oil may lead to their destruction by dissolving theconstituents of liposomes in the oil. To investigate this question,rabbits (2 rabbits in each group) are immunized with the vaccine (100%oil formulation) that is stored for up to 5 months at 5° C. and −20° C.Storage of the vaccine at 5° C. for 5 months reduced anti-SIZP titers ofrabbits by only 28% (Table 6C; P=0.002; F(5,33)=4.9). Storage of thevaccine at −20° C. for 5 months reduced anti-SIZP titers by only 14%(Table 6D; P=<0.001; F(5,35)=23.7). These results indicate that mostliposomes remain intact in oil since immunization of rabbits with asingle injection of SIZP suspended in Freund's complete adjuvant withoutliposome encapsulation results in low titers (Table 6E). TABLE 6C Effectof storage of the vaccine composition with 100% oil formulation on theproduction of anti-SIZP antibodies by rabbits Anti-SIZP titer¹(% ofreference serum) Storage² Post-immmunization (months) (months) 0 1 2 3 45 6 Average SE 0 0 60 123 112 163 109 119 114 11.2 0 26 108 112 163 131141 1 0 26 112 183 121 122 100 113 11.9 0 77 133 142 117 70 153 2 0 63176 128 70 127 57 98 13.9 0 50 110 100 ND ND ND 3 0 23 138 168 104 117143 94 13.3 0 35 100 113 76 63 42 4 0 17 39 104 63 73 100 68 10.1 0 47136 73 26 45 85 5 0 22 127 69 66 140 99 82 11.8 0 27 111 57 54 140 74¹The vaccine composition (100% oil formulation) is placed in biobulletspurchased from Ballistivet ™ and surgically implanted intramuscularlyinto rabbits.²Biobullets are stored at 5° C.

TABLE 6D Effect of storage of the vaccine composition with 100% oilformulation on the production of anti-SIZP antibodies by rabbitsAnti-SIZP titer¹ (% of reference serum) Storage² Post-immunization(months) (months) 0 1 2 3 4 5 6 Average SE 0 0 60 123 112 163 109 119114 11.2 1 26 108 112 163 131 141 1 0 9 13 13 41 19 60 27 6.7 0 6 16 1473 33 ND 2 0 9 14 23 70 136 116 51 12.7 0 6 30 19 41 95 57 3 0 13 23 127100 73 88 57 11.1 0 13 18 90 61 58 30 4 0 13 68 50 71 80 122 85 14.0 013 114 134 81 100 179 5 0 9 114 75 87 146 92 98 13.7 1 22 108 116 109179 125¹The vaccine composition (100% oil formulation) is placed in biobulletspurchased from Ballistivet ™ and surgically implanted intramuscularlyinto rabbits.²Biobullets are stored at −20° C.

TABLE 6E Production of anti-SIZP antibodies by rabbits immunized with asingle administration of SIZP without encapsulation of SIZP in liposomesAnti-SIZP titer (% of reference serum)¹ Rabbit ID Post-immunization(months) No. 0 1 2 3 4 1 0 43 19 9 2 2 0 27 7 4 1¹Rabbits are immunized with a single administration of SIZP (50μg/rabbit) suspended in Freund's Complete Adjuvant.

To determine if the vaccine formulated to contain no aqueous phase wouldresult in a good response in another mammalian species, grey seals(Halichoerus grypus) are immunized with the vaccine containing eitherequal oil and aqueous phases, only an aqueous phase, or only an oilphase (Table 6F). There was no difference in anti-SIZP titers in thevaccine that contained equal oil and aqueous phases or only oil butadministration of the vaccine that contained all ingredients except oil,resulted in significantly lower titers. TABLE 6F Effect of oil contentof the vaccine composition on the production of anti-SIZP antibodies bygrey seals. Oil Anti-SIZP titer (% of reference serum)¹ contentPost-immunization (months) (%, v/v) 0 1 2 3 4 0² 0 5 4 1 1 0 1 2 1 1 50³0 7 9 145 107 0 41 52 82 38 100⁴ 0 20 28 79 60 0 3 30 90 50¹Each line presents titers of blood samples taken from the same greyseal.²Liposomes containing SIZP (100 μg/grey seal) and heat killedMycobacterium tuberculosis (2 mg/grey seal), the active ingredient inFreund's complete adjuvant as supplied by Sigma Chemical Co. and used inall studies reported herein are suspended in saline (0.5 ml).³Liposomes containing SIZP (100 μg/grey seal) are suspended in saline(0.5 ml) and this aqueous phase is emulsified in Freund's completeadjuvant (0.5 ml).⁴Liposomes containing SIZP (100 μg/grey seal) are freeze-dried and theresulting freeze-dried liposomes suspended in Freund's complete adjuvant(0.5 ml).

Example 7 Use of Archaebacterial Lipids in Liposomes

Liposomes are completely closed lipid membranes that can be made from avariety of lipid materials. In this example, liposomes made usingarchaebacterial lipids are compared to liposomes made using soybeanlecithin for their ability to stimulate antibody production by rabbits(Table 7). Liposomes made with soybean lecithin result in betterproduction of anti-SIZP antibodies than liposomes made witharchaebacterial lipids. TABLE 7 Production of anti-SIZP antibodies byrabbits immunized with liposomes prepared with archaebacterial lipids orsoybean lecithin. Anti-SIZP titer (% of reference serum) Type of Postimmunization (months) ID NO. lipid 0 1 2 3 4 5 112 Soybean 0 143 195 137197 98 lecithin 115 Soybean 0 200 198 157 179 106 lecithin 117Archaebacterial 0 46 30 37 13 ND lipids 118 Archaebacterial 0 7 4 8 2 NDlipidsND = not determined.

Example 8 Immunization Against Streptokinase

The vaccine composition is unique in producing high titers of anti-SIZPantibodies that are long-lasting following a single administration. Todetermine if the vaccine composition would produce high antibody titerswith other antigens, rabbits are immunized with streptokinase.

Streptokinase is an exoprotein produced by pathogenic strains of theStreptococci family of bacteria. As an activator of vascularfibrinolysis its therapeutic usefulness has been appreciated for manyyears in the treatment of myocardial infarction. Streptokinase unfoldsin a non-cooperative manner. Therefore, the protein can assume a numberof partially folded states that contain some regions that appear to benative and others that are unfolded. Three domains of differentstability exist that are independent of other regions of the protein(Teuten et al., 1993, Biochem. J. 290:313-319). Native streptokinasecontains immunodominant epitopes in the C-terminal region (Torrens etal., 1999, Immunology Letters 70:213-218). The C-terminal region isrelatively unstructured (Parrado et al., 1996, Protein Sci 5:693-704)therefore heat treatment cannot alter the structure since it wasunstructured before heat treatment. The thermal stability of domain C issignificantly increased by its isolation from the rest of the chain(Connejero-Lara et al., 1996, Protein Sci 5:2583-2591). Loss of theC-terminal region results in a less immunogenic protein but does exposeimmunogenic epitopes hidden in the native molecule. In our studies, theC-terminal region was present in native and heat-treated streptokinase,therefore, as the immunodominant region of the protein, it woulddetermine the response of the rabbits. If the C-terminal region retainedthe same epitopes following heat treatment as found in the native state,one would not expect to find a difference in binding ofanti-streptokinase antibodies to native and heat-treated streptokinase.These are exactly the observations found (Table 8). We have proposedthat delivery of denatured proteins using a vaccine composition of thepresent invention favours the production of antibodies directed againstnative epitopes. This is supported by the studies of alcoholdehydrogenase in Example 2. The results with streptokinase areconsistent with this proposal since heat treatment would not alter thestructure of the immunodominant region and the prediction follows thatthere would be no difference in the immune response of rabbits beingimmunized with native and heat treated streptokinase regardless of thedelivery system employed. These are precisely our observations (Table8). TABLE 8 Epitope mapping of rabbit anti-streptokinase sera fromrabbits immunized with native and heat-treated streptokinase (100° C.for 10 minutes in 5% mercaptoethanol) using conventional immunizationprotocols¹ or the method of the present invention². Titer (% ofreference serum)³ Native Heat-treated Immunization streptokinasestreptokinase Rabbit Post-immunization (months) ID Antigen Delivery 0 12 0 1 2 21 Native Invention 0 100 122 0 98 122 24 Native Invention 0 8298 0 53 108 25 Native Conventional 0 10 89 0 8 100 20 NativeConventional 0 9 94 0 3 92 23 Heat- Invention 0 10 34 0 15 31 treated 28Heat- Invention 0 25 99 0 24 94 treated 27 Heat- Conventional 0 9 107 07 94 treated 30 Heat- Conventional 0 10 100 0 8 94 treated¹Rabbits were immunized with 75 μg streptokinase in Freund's completeadjuvant followed by one booster injection one month later with 75 μgstreptokinase in Freund's incomplete adjuvant.²Rabbits were immunized with a single injection of 75 μg streptokinasein a vaccine of the present invention.³Titers were measured with both native and heat-treated streptokinase.

It is evident from the results that a single injection of streptokinaseusing a vaccine of the present invention produced anti-streptokinasetiters similar to titers obtained by the conventional primary andbooster injection protocols. Also, regardless of the immunizationprotocol used, that is the present invention or conventional, theantibodies produced bound to native and heat-treated streptokinaseequally well.

Example 9 Use of an Edible Vegetable Oil

A vaccine composition was formulated in accordance with this inventionusing Canola oil in place of mineral oil. The results are shown in Table9. The results indicate that vaccines formulated with Canola oil produceanti-SIZP antibodies in rabbits, therefore, Canola oil is useful.However, the titer levels are not as high as with mineral oil. TABLE 9Effect of Canola oil on production of anti-SIZP antibodies in rabbitsAnti-SIZP titer (% of reference serum) Rabbit Vaccine Post-immunization(months) ID formulation 0 1 2 3 4 5 6 7 4 Alum/mineral oil 0 111 123 105124 95 125 157 14 0 231 132 205 178 279 258 251 9 FCA/mineral oil¹ 0 162264 310 ND ND ND ND 26 0 351 166 112 ND ND ND ND 34 FCA/Canola oil² 0 2724 37 ND ND ND ND 36 0 18 24 36 ND ND ND ND¹Freund's complete adjuvant (FCA) was obtained from a commercial source(Sigma) and was formulated with mineral oil.²FCA/Canola oil contained the same quantity of Mycobacterium heat-killedcells as present in FCA but the mineral oil component of FCA wasreplaced with edible Canola oil.ND = not determined

Example 10 Immunization Against Hepatitis B

A hepatitis B vaccine was formulated in accordance with the presentinvention using 5 micrograms hepatitis B surface antigen (RecombivaxHB™, a recombinant hepatitis B antigen) containing alum adjuvantencapsulated in liposomes containing soybean lecithin (0.05 g) andcholesterol (0.005 g) suspended in saline (0.25 ml) then emulsified inlow viscosity mineral oil (o.225 ml) and mannide oleate (0.025 ml). Aconventional hepatitis B vaccine using 5 micrograms hepatitis B surfaceantigen (Recombivax HB™) containing alum adjuvant in a volume of 0.5 mlaqueous medium as recommended by the manufacturer was also administered.Eight rabbits were immunized with the vaccine prepared in accordancewith the present invention and eight rabbits were immunized with theconventional vaccine. Results are shown in Table 10.

It is evident from Table 10 that the vaccine prepared in accordance withthe present invention results in about 6 times more antibody 1 monthpost-immunization than conventional delivery of hepatitis B surfaceantigen. TABLE 10 Production of anti-HepB antibodies by rabbitsimmunized with a commercial HepB vaccine or with a HepB vaccineformulated in accordance with the present invention Anti-HepB titer(mlU/ml)¹ Rabbit Post-immunization (months) ID 0 1 Commercial vaccine 960 736 101 0 1237 97 0 488 100 0 1877 99 0 6251 103 0 8384 98 0 688 102 01568 Average 0 2654 Vaccine of the invention 93 0 32,341 95 0 3371 88 05717 81 0 23,808 83 0 9344 84 0 17,856 79 0 9344 85 0 21,675 Average 015,432¹Antibody titers were measured using the enzyme immunoassay for thedetection of antibody to hepatitis B surface antigen (anti-HBs)distributed by DiaSorin Inc., Stillwater, MN, USA.

Example 11 Effect of Formulating Vaccines with Alum Adjuvant Inside andOutside of Liposomes

Vaccines were prepared as follows: Group SIZP antigen Alum Medium 1inside liposome inside liposome saline 2 inside liposome outsideliposome saline 3 inside liposome inside liposome oil 4 inside liposomeoutside liposome oil 5 control - no liposomes oil 6 control - noliposomes saline

Groups 1-4 were prepared with 100 μg SIZP encapsulated in liposomesformed with 0.1 g soybean lecithin and 0.01 g cholesterol. The liposomesin Groups 1 and 3 also contained 100 μl ImjectAlum™. In Groups 2 and 4,100 μl ImjectAlum™ was placed outside the liposomes. In Groups 1 and 2,the liposomes were suspended in 0.25 ml saline and this suspensionemulsified in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate. In Groups 3 and 4, the liposomes were freeze dried thensuspended in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate and this suspension emulsified in 0.25 ml saline. In Group 5, 100μg SIZP and 100 μl ImjectAlum™ were freeze dried, then suspended in0.225 ml low viscosity mineral oil and 0.025 ml mannide oleate andemulsified in 0.25 ml saline. In Group 6, 100 μg SIZP and 100 μlImjectAlum™ were freeze dried, then suspended in 0.25 ml saline andemulsified in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate. Rabbits were immunized with the six groups of vaccines and theresults are shown in Table 11. TABLE 11 Production of anti-SIZPantibodies by rabbits immunized with four formulations of a vaccine ofthe present invention containing alum adjuvant (Groups 1-4) and twocontrol formulations containing alum adjuvant (Groups 5-6) Anti-SIZPtiter (% reference serurm) Average titer Standard Post-immunizationPost-immunization Error of Rabbit months months average titer Group ID 01 2 3 0 1 2 3 1 2 3 1 49 2 107 176 183 0 135 203 236 22 28 37 1 76 0 134105 124 1 71 0 70 249 331 1 82 0 182 236 268 1 78 0 182 249 274 2 73 0373 273 304 0 287 207 258 32 24 15 2 42 0 328 199 281 2 62 0 259 194 2442 77 0 182 131 235 2 74 0 295 238 225 3 63 0 363 135 113 0 300 171 16032 26 34 3 67 0 286 175 140 3 70 0 332 241 261 3 80 0 218 131 125 4 48 0383 210 113 0 200 138 108 49 20 12 4 64 0 129 109 121 4 61 0 125 98 63 460 0 148 140 115 4 66 0 215 134 130 5 45 0 21 133 120 0 26 96 123 8 2933 5 65 0 6 26 42 5 69 0 49 110 121 5 72 0 12 36 89 5 50 0 40 176 242 668 0 28 31 22 0 16 22 18 4 3 1 6 75 0 9 16 16 6 46 0 18 23 17 6 43 0 1019 16

Example 12 Effect of Formulating Vaccines with Heat Killed Mycobacteriumtuberculosis Adjuvant Inside and Outside of Liposomes

Vaccines were prepared as follows: Heat-killed Group SIZP antigen M.tuberculosis Medium 1 inside liposome inside liposome saline 2 insideliposome outside liposome saline 3 inside liposome inside liposome oil 4inside liposome outside liposome oil

Groups 1-4 were prepared with 100 μg SIZP encapsulated in liposomesformed with 0.1 g soybean lecithin and 0.01 g cholesterol. The liposomesin Groups 1 and 3 also contained 200 μg heat killed M. tuberculosis. InGroups 2 and 4, 200 μg heat killed M. tuberculosis was placed outsidethe liposomes. In Groups 1 and 2, the liposomes were suspended in 0.2 mlsaline and this suspension emulsified in 0.18 ml low viscosity mineraloil and 0.02 ml mannide oleate. In Groups 3 and 4, the liposomes werefreeze dried then suspended in 0.18 ml low viscosity mineral oil and0.02 ml mannide oleate and this suspension emulsified in 0.2 ml saline.Rabbits were immunized with the four groups of vaccines and the resultsare shown in Table 12. TABLE 12 Production of anti-SIZP antibodies byrabbits immunized with four formulations of a vaccine of the presentinvention containing heat killed M. tuberculosis Anti-SIZP titerStandard (% reference serum) Average titer Error Of Post-immunizationPost-immunization average Rabbit months months titer Group ID 0 1 2 3 01 2 3 1 2 3 1 33 0 245 236 259 0 318 262 437 51 18 126 1 29 0 390 288614 2 32 0 544 515 633 0 576 532 638 22 12 3 2 22 0 608 549 642 3 9 0162 264 310 0 293 599 508 93 237 140 3 13 0 424 933 707 4 16 0 454 276532 0 403 221 322 36 39 149 4 26 0 351 166 112

Example 13 Immunization of Rabbits with Native and Denatured YeastAlcohol Dehydrogenase (ADH) Together with Alum Adjuvant

Vaccines of the present invention were formulated containing 100 μg ofnative or denatured ADH together with 100 μl ImjectAlum™ encapsulated inliposomes formed with 0.1 g soybean lecithin and 0.01 g cholesterol. Theliposomes were suspended in 0.25 ml saline and the suspension emulsifiedin 0.225 ml low viscosity mineral oil and 0.025 ml mannide oleate.

Conventional vaccines were formulated containing 100 μg of native ordenatured ADH together with 100 μl ImjectAlum™ and suspended in 0.5 mlsaline.

Denatured ADH was prepared by heating ADH to 100° C. for 30 minutes andtreating with 10% mercaptoethanol for 30 minutes at room temperature tocleave disulfide bonds. Mercaptoethanol was removed by dialysis for 12hours and denatured ADH was recovered by freeze drying.

Results comparing the vaccines of the present invention to theconventional vaccines are shown in Table 13. TABLE 13 Production ofanti-ADH antibodies by rabbits immunized with native and denatured ADHusing alum as adjuvant Anti-ADH titer (% reference serum)Post-immunization (months) Rabbit Delivery Native ADH Denatured ADH IDSystem 0 1 2 3 0 1 2 3 Native ADH 54 invention 0 24 42 66 0 8 4 4 57 0100 143 146 0 29 3 6 55 0 91 85 111 0 26 3 5 40 conventional 0 1 1 2 0 10 1 44 0 1 1 1 0 1 0 1 47 0 5 4 8 0 3 1 1 Denatured ADH 53 invention 0 12 5 0 1 1 2 58 0 1 1 2 0 1 0.5 1 52 0 1 4 15 0 1 0.5 1 51 conventional 02 4 4 0 1 0.5 2 59 0 1 1 4 0 1 0.5 0 56 0 2 2 2 0 1 0.5 0

The results show that delivery of native or denatured ADH using aformulation of the present invention results in an increased productionof anti-ADH antibodies compared to the production of anti-ADH antibodiesby rabbits immunized against native or denatured ADH using conventionalmethods. Furthermore, rabbits immunized with denatured ADH produced moreantibodies directed against native ADH when a formulation of the presentinvention is used rather than when denatured ADH is delivered byconventional means with no booster injections.

Example 14 Epitope Mapping

Epitope mapping experiments to demonstrate that vaccines of the presentinvention produce antibodies having different binding specificity for anantigen than achieved by conventional immunization protocols of primaryand secondary booster injections. Fragments of the ZP antigen werespecifically used but it is expected that other antigens will behave ina similar manner.

Conventional immunization of grey and harp seals with a primaryinjection and two booster injections results in low anti-SIZP antibodytiters that peak two months post-immunization in both grey and harpseals. In contrast, immunization with a vaccine formulated in accordancewith the present invention produces anti-SIZP antibody titers thatpersist for at least 24 months in grey seals and 5-6 months in harpseals, with one exception. Titers in harp seals reach a plateau thatpersist for 6-10 months post-immunization. Therefore, a vaccineformulated in accordance with the present invention induces highanti-SIZP titers with long duration compared to conventionalimmunization protocols using primary and booster injections.

The polypeptide fragments of ZPB and ZPC that were used in epitopemapping to demonstrate that anti-SIZP antibodies produced followingconventional immunization protocols have a different binding specificitythan anti-SIZP antibodies produced following immunization with a vaccineof the present invention are shown in FIG. 1. The fragments ZPB1, ZPB2,ZPC1 and ZPC2 are short length polypeptides and do not have thethree-dimensional structures of full length ZPB and ZPC. In FIG. 1, thefull-length unprocessed polypeptides are shown above the two ZPB and ZPCfragments. The secretory signal peptides that are cleaved in the nativeproteins are shaded in black.

Anti-SIZP grey seal antibodies produced following conventionalimmunization (a primary and two booster injections using FCA adjuvant)have a high affinity for the ZPB1, ZPB2, ZPC1 and ZPC2 fragments (Table14A, seal ID 1). In contrast, grey seals immunized with a vaccineformulated in accordance with the present invention (Table 14A, seal ID76 and 96) produce antibodies that have a low affinity for fragmentsZPB2, ZPC1 and ZPC2 one year post-immunization and low affinity for allfour fragments three years post-immunization. The four fragmentstogether account for 80% of the protein found in SIZP. TABLE 14A Epitopemapping of grey seal anti-SIZP antibodies using recombinant fragments ofZPB and ZPC produced in E. coli Post- Seal immunization Binding relativeto SIZP (%) ID (months) ZPB1 ZPB2 ZPC1 ZPC2 Total 1 3 30 44 59 41 174 14 71 70 83 63 287 76 12 54 25 8 12 99 76 36 18 9 13 11 51 96 12 47 18 1510 90 96 36 10 8 15 10 43

A temporal study of the binding specificity of antibodies produced bygrey seals immunized with a vaccine formulated in accordance with thepresent invention indicated that antibodies produced earlypost-immunization (<7 months) bind to epitopes found predominantly onthe ZPB1 fragment. Antibodies produced late post-immunization (>7months) have lower affinity for ZPB1 and the other three fragments.ZPB1, ZPB2, ZPC1 and ZPC2 are low molecular weight and are notglycosylated. These fragments have less three-dimensional structure thanfull-length ZPB and ZPC because of their low molecular weight.Therefore, antibodies that bind to SIZP but not ZPB1, ZPB2, ZPC1 or ZPC2must either be recognizing three-dimensional structures found only onfull-length ZPB and ZPC or the carbohydrate covalently linked to theseproteins. Since the total amount of antibody bound to the fragmentsearly post-immunization exceeds or is equivalent to the amount ofantibody binding to ZPB and ZPC, carbohydrate-recognizing antibody musthave a minor role. This implies that 3-D structures determine thedifference in binding to the fragments as opposed to SIZP. A survey ofnine other grey seals immunized with SIZP in a vaccine of the presentinvention indicates similar reduction to antibodies produced 5 months ormore post-immunization.

In another experiment, three of four rabbits immunized with a vaccine ofthe present invention produced antibodies with a higher affinity forepitopes in ZPB1 than in the other three fragments. Only 20-40% of theantibodies produced by all four rabbits bound to epitopes found in thefour ZP fragments. Therefore, 60-80% of the anti-SIZP antibodiesproduced by rabbits immunized with a vaccine of the present inventionbound only to epitopes found in full length ZPB and ZPC. Therefore,60-80% of antibodies produced in rabbits immunized with a vaccine of thepresent invention recognize epitopes related to native 3-D structures.

In yet another experiment, immunization of harp seals (156 and 162) byconventional protocols of a primary injection using FCA adjuvantfollowed by booster injections with FIA adjuvant produced antibodiesearly post-immunization that bound to epitopes found in ZPB1, ZPB2 andZPC2 (harp seal 156) or all four fragments (harp seal 162) as well as inSIZP (Table 14B). In contrast, immunization of harp seal 151 with avaccine formulated in accordance with the present invention producedantibodies early post-immunization (<5 months) that bound to epitopesfound in all four ZP fragments but antibodies produced latepost-immunization (>7 months) bound to epitopes found only on fulllength ZPB and ZPC (Table 14B). Only 30-40% of the antibodies producedby immunization of harp seal 153 with a vaccine of the present inventionbound well to epitopes on the four ZP fragments, implying that 60-70% ofthe antibodies produced by harp seal 153 during the 7 monthpost-immunization period bound only to epitopes found in full length ZPBand ZPC. These epitopes must be related to structures found only in fulllength ZPB and ZPC implying 3-D structures. Immunization of hooded seal1 with a vaccine of the present invention produced antibodies with asimilar temporal sequence of specificity as harp seal 151. TABLE 14BEpitope mapping of harp and hooded seal anti-SIZP antibodies usingrecombinant fragments of ZPB and ZPC produced in E. coli Post-immunization Binding relative to SIZP (%) Seal ID (months) ZPB1 ZPB2ZPC1 ZPC2 Total Harp 156 2 9 8 7 7 31 3 30 19 10 24 83 4 36 38 2 34 110Harp 162 2 8 10 11 11 40 3 33 26 19 24 102 Harp 151 1 40 33 36 33 142 341 28 32 21 122 5 21 27 35 34 117 6 50 26 30 44 150 7 8 6 9 8 31 9 4 1732 10 63 Harp 153 2 12 6 11 9 38 3 16 12 9 12 49 4 6 5 7 3 21 5 13 8 1211 44 6 12 9 11 12 44 7 9 6 0 7 22 Hooded 1 2 30 22 26 27 105 3 33 25 2830 116 4 37 32 38 34 141 5 31 24 29 31 115 7 15 12 12 11 50 8 12 13 11 844

1-17. (canceled)
 18. A method for potentiating an immune response in ananimal, which method comprises administering to the animal an effectiveamount of a vaccine composition comprising: (a) a carrier comprising acontinuous phase of a hydrophobic substance; (b) liposomes; (c) anantigen encapsulated in said liposomes, said antigen being an antigenwhich when not in said vaccine composition has, a conformation otherthan its native conformation, with the proviso that said antigen isother than a zona pellucida-derived antigen; and (d) a suitableadjuvant.
 19. The method of claim 18, wherein the hydrophobic substancesis a liquid.
 20. The method of claim 18, wherein the carrier is an oilor a water-in-oil emulsion.
 21. The method of claim 20, wherein the oilis mineral oil, a vegetable oil or a nut oil.
 22. The method of claim21, wherein the adjuvant is alum or another compound of aluminum
 23. Themethod of claim 21, wherein the antigen is alcohol dehydrogenase, or ahepatitis B antigen
 24. The method of claim 20, wherein the antigenelicits an antibody that recognizes a native epitope.
 25. The method ofclaim 24, wherein the native epitope is a mammalian epitope.
 26. Themethod of claim 25, wherein the mammal is a horse, a rabbit, a deer or acat.
 27. The method of claim 20, wherein the composition issubstantially free of lipid A. 28-43. (canceled)
 44. The method of claim22, wherein the adjuvant is alum.
 45. The method of claim 20, whereinthe antigen is a non-native, recombinant or denatured protein, arecombinant or synthetic peptide, or a fragment thereof.
 46. The methodof claim 45, wherein the antigen is a viral, bacterial, protozoal ormammalian antigen.
 47. The method of claim 20, wherein the liposomescomprise unesterified cholesterol and a phospholipid with at least onehead group selected from the group consisting of phosphoglycerol,phosphoethanolamine, phosphoserine, phosphocholine and phosphoinositol.48. The method of claim 18, wherein said vaccine composition isadministered parenterally.
 49. The method of claim 18, wherein saidanimal is a horse, rabbit, deer or cat.